Ubiquitin regulatory elements

ABSTRACT

The present invention provides compositions and methods for regulating expression of heterologous nucleotide sequences in a plant. Compositions are novel nucleotide sequences for a constitutive regulatory element isolated from sorghum. A method for expressing a heterologous nucleotide sequence in a plant using the regulatory sequences disclosed herein is provided. The method comprises transforming a plant cell to comprise a heterologous nucleotide sequence operably linked to one or more of the regulatory sequences of the present invention and regenerating a stably transformed plant from the transformed plant cell.

CROSS REFERENCE

This utility application claims the benefit U.S. Provisional ApplicationNo. 61/092,205 filed Aug. 27, 2008, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more particularly to regulation of gene expression in plants.

BACKGROUND OF THE INVENTION

As the field of plant bioengineering develops and more genes becomeaccessible, a greater need exists for transforming with multiple genes.These multiple exogenous genes typically need to be controlled byseparate regulatory sequences. Some genes should be regulatedconstitutively whereas other genes should be expressed at certaindevelopmental stages or location in the transgenic organism.Accordingly, a variety of regulatory sequences having diverse effects isneeded.

In addition, undesirable biochemical interactions can result from usingthe same regulatory sequence to control more than one gene. For example,transformation with multiple copies of a regulatory element may causehomologous recombination between two or more expression systems,formation of hairpin loops caused from two copies of the same promoteror enhancer in opposite orientation in close proximity, competitionbetween identical expression systems for binding to commonpromoter-specific regulatory factors, and inappropriate expressionlevels of an exogenous gene due to trans effects of a second promoter orenhancer.

In view of these considerations, a goal in this field has been thedetection and characterization of new regulatory sequences fortransgenic control of DNA constructs.

Isolation and characterization of constitutive promoters and terminatorsthat can serve as regulatory elements for expression of isolatednucleotide sequences of interest in a constitutive manner are needed forimproving traits in plants.

SUMMARY OF THE INVENTION

The invention is directed to a promoter from a Sorghum bicolorubiquitin-encoding gene, useful as a regulatory region and providing forconstitutive expression of an operably linked nucleotide sequence. Theinvention is further directed to functional fragments which function todrive constitutive expression of operably linked nucleotide sequences.Expression cassettes having the nucleotide sequence, plants expressingsame and methods of use in driving expression of operably linkednucleotides sequences are within the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: The nucleotide sequence of the sorghum ubiquitin regulatoryelement (SEQ ID NO: 1). The promoter (SEQ ID NO: 2) is the first 831nucleotides, with the intron underlined (SEQ ID NO: 3).

FIG. 2: A comparison of the sorghum ubiquitin regulatory element(promoter and intron) of the invention (SEQ ID NO: 1) with maizeubiquitin promoter region (promoter and intron) (SEQ ID NO: 4).

DETAILED DESCRIPTION OF THE INVENTION

All references referred to are incorporated herein by reference. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting.

Expression of heterologous DNA sequences in a plant host is dependentupon the presence of operably linked regulatory elements that arefunctional within the plant host. Choice of the regulatory element willdetermine when and where within the organism the heterologous DNAsequence is expressed. Where continuous expression is desired throughoutthe cells of a plant and/or throughout development, constitutivepromoters are utilized. In contrast, where gene expression in responseto a stimulus is desired, inducible promoters are the regulatory elementof choice. Where expression in specific tissues or organs are desired,tissue-specific promoters may be used. That is, they may driveexpression in specific tissues or organs. Such tissue-specific promotersmay be temporally constitutive or inducible. In either case, additionalregulatory sequences upstream and/or downstream from a core promotersequence may be included in expression constructs of transformationvectors to bring about varying levels of expression of heterologousnucleotide sequences in a transgenic plant.

In accordance with the invention, nucleotide sequences are provided thatallow regulation of transcription in a constitutive manner. Thus, thecompositions of the present invention comprise novel nucleotidesequences for plant regulatory elements natively associated with thenucleotide sequences coding for Sorghum bicolor ubiquitin protein,herein identified as SB-UBI.

Ubiquitin is a polypeptide found in all eukaryotes and has been studiedfor its role in a wide range of cellular functions. Promoters of theubiquitin gene have been isolated. For example, in U.S. Pat. Nos.5,510,474, 5,614,399, 6,054,574 and 6,020,190 to Quail is describedubiquitin promoters which include a heat shock element and intron.Jilka, et al., describe another maize ubiquitin type promoter at U.S.Pat. No. 6,977,325. Xia, et al., identified a soybean genomic clonecontaining a ubiquitin gene (Xia, et al., (1994) Plant Physiol.104:805-806). These sequences are reported at GenBank accession numbersD16248.1 and D2823.1. Also, Finer, et al., have discussed analysis of asoybean ubiquitin promoter, but did not provide a sequence (Finer, etal., (2006) “Characterization of soybean promoters through evaluation ofGFP expression in transgenic soybean” The 11^(th) Biennial Conference onthe Molecular and Cellular Biology of the Soybean, Aug. 5-8, 2006,University of Nebraska, Lincoln, Nebr.).

In an embodiment, the regulatory element drives transcription in aconstitutive manner, wherein said regulatory element comprises anucleotide sequence selected from the group consisting of: a) sequencesnatively associated with, and that regulate expression of DNA coding forsorghum SB-UBI (Sorghum bicolor ubiquitin protein); b) the nucleotidesequence set forth in SEQ ID NO: 1, 2 or 3 or c) a sequence comprising afragment of the nucleotide sequence set forth in either of SEQ ID NO: 1,2 or 3.

Further embodiments are to expression cassettes, transformation vectors,plants, plant cells and plant cells comprising the above nucleotidesequences. The invention is further to methods of using the sequence inplants and plant cells.

A method for expressing an isolated nucleotide sequence in a plant usingthe regulatory sequences disclosed herein is provided. The methodcomprises transforming a plant cell with a transformation vector thatcomprises an isolated nucleotide sequence operably linked to one or moreof the plant regulatory sequences of the present invention andregenerating a stably transformed plant from the transformed plant cell.In this manner, the regulatory sequences are useful for controlling theexpression of endogenous as well as exogenous products in a constitutivemanner.

Alternatively, it might be desirable to inhibit expression of a nativeDNA sequence within a plant's tissues to achieve a desired phenotype. Inthis case, such inhibition might be accomplished with transformation ofthe plant to comprise a tissue-specific promoter operably linked to anantisense nucleotide sequence, such that tissue-specific expression ofthe antisense sequence produces an RNA transcript that interferes withtranslation of the mRNA of the native DNA sequence in a subset of theplant's cells.

Under the regulation of the regulatory element will be a particularpolynucleotide sequence of interest. Expression of the sequence ofinterest will provide for modification of the phenotype of the plant.Such modification includes modulating the production of an endogenousproduct, as to amount, relative distribution or the like or productionof an exogenous expression product to provide for a novel function orproduct in the plant.

By “constitutive” is intended expression which is capable oftranscribing operatively linked DNA sequences efficiently and expressingsaid DNA sequences in multiple tissues.

By “regulatory element” is intended sequences responsible expression ofthe associated coding sequence including, but not limited to, promoters,terminators, enhancers, introns and the like.

By “promoter” is intended a regulatory region of DNA capable ofregulating the transcription of a sequence linked thereto. It usuallycomprises a TATA box capable of directing RNA polymerase II to initiateRNA synthesis at the appropriate transcription initiation site for aparticular coding sequence.

A promoter may additionally comprise other recognition sequencesgenerally positioned upstream or 5′ to the TATA box, referred to asupstream promoter elements, which influence the transcription initiationrate and further include elements which impact spatial and temporalexpression of the linked nucleotide sequence. It is recognized thathaving identified the nucleotide sequences for the promoter regiondisclosed herein, it is within the state of the art to isolate andidentify further regulatory elements in the 5′ region upstream from theparticular promoter region identified herein. Thus the promoter regiondisclosed herein may comprise upstream regulatory elements such as thoseresponsible for tissue and temporal expression of the coding sequence,and may include enhancers, the DNA response element for atranscriptional regulatory protein, ribosomal binding sites,transcriptional start and stop sequences, translational start and stopsequences, activator sequence and the like.

In the same manner, the promoter elements which enable constitutiveexpression can be identified, isolated and used with other corepromoters to confirm constitutive expression. By core promoter is meantthe minimal sequence required to initiate transcription, such as thesequence called the TATA box which is common to promoters in genesencoding proteins. Thus the upstream promoter of SB-UBI can optionallybe used in conjunction with its own or core promoters from othersources. The promoter may be native or non-native to the cell in whichit is found.

The isolated promoter sequence of the present invention can be modifiedto provide for a range of expression levels of the isolated nucleotidesequence. Less than the entire promoter region can be utilized and theability to drive constitutive expression retained. It is recognized thatexpression levels of mRNA can be modulated with specific deletions ofportions of the promoter sequence. Thus, the promoter can be modified tobe a weak or strong promoter. Generally, by “weak promoter” is intendeda promoter that drives expression of a coding sequence at a low level.By “low level” is intended levels of about 1/10,000 transcripts to about1/100,000 transcripts to about 1/500,000 transcripts. Conversely, astrong promoter drives expression of a coding sequence at a high level,or at about 1/10 transcripts to about 1/100 transcripts to about 1/1,000transcripts. Generally, at least about 20 nucleotides of an isolatedpromoter sequence will be used to drive expression of a nucleotidesequence.

It is recognized that to increase transcription levels enhancers can beutilized in combination with the promoter regions of the invention.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are known in the art and include theSV40 enhancer region, the 35S enhancer element and the like.

The promoter of the present invention can be isolated from the 5′ regionof its native coding region or 5′ untranslated region (5′ UTR). Likewisethe terminator can be isolated from the 3′ region flanking itsrespective stop codon.

The term “isolated” refers to material, such as a nucleic acid orprotein, which is: (1) substantially or essentially free from componentswhich normally accompany or interact with the material as found in itsnaturally occurring environment or (2) if the material is in its naturalenvironment, the material has been altered by deliberate humanintervention to a composition and/or placed at a locus in a cell otherthan the locus native to the material. Methods for isolation of promoterregions are well known in the art. One method is the use of primers andgenomic DNA used in conjunction with the Genome Walker Kit™ (Clonetech).

The SB-UBI promoter region set forth in SEQ ID NO: 1 is 1899 nucleotidesin length and includes the promoter of SEQ ID NO: 2 which is 831nucleotides in length and and intron of SEQ ID NO: 3 which is 1068nucleotides in length. The SB-UBI promoter was isolated from the sorghumbicolor SB-UBI coding region. It was isolated by identifying the sorghumhomolog of the maize ubiquitin, then examining a collection of sorghumgenomic sequence and looking for hits. Once a hit was identified, thesequence upstream of the open reading frame was used to design PCRprimers to amplify a product similar in length to the maize ubiquitinpromoter.

The regulatory regions of the invention may be isolated from any plant,including, but not limited to sorghum (Sorghum bicolor, Sorghumvulgare), corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.),alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale),sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean(Glycine max), tobacco (Nicotiana tabacum), millet (Panicum spp.),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), oats (Avena sativa), barley (Hordeum vulgare),vegetables, ornamentals and conifers. Preferably, plants include corn,soybean, sunflower, safflower, canola, wheat, barley, rye, alfalfa andsorghum.

Maize promoters have been used repeatedly to drive expression of genesin non-maize plants, including tobacco (Yang and Russell, (1990);Geffers, et al., (2000); Vilardell, et al., (1991)), cultured rice cells(Vilardell, et al., (1991)), wheat (Oldach, et al., (2001);Brinch-Pedersen, et al., (2003)), rice (Cornejo, et al., (1993);Takimoto, et al., (1994)), sunflower (Roussell, et al., (1988)) andprotoplasts of carrot (Roussell, et al., (1988)).

Regulatory sequences from other plants may be isolated according towell-known techniques based on their sequence homology to the homologouscoding region of the coding sequences set forth herein. In thesetechniques, all or part of the known coding sequence is used as a probewhich selectively hybridizes to other sequences present in a populationof cloned genomic DNA fragments (i.e. genomic libraries) from a chosenorganism. Methods are readily available in the art for the hybridizationof nucleic acid sequences. An extensive guide to the hybridization ofnucleic acids is found in Tijssen, Laboratory Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays”, Elsevier, New York (1993) and CurrentProtocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995).

“Functional variants” of the regulatory sequences are also encompassedby the compositions of the present invention. Functional variantsinclude, for example, the native regulatory sequences of the inventionhaving one or more nucleotide substitutions, deletions or insertions.Functional variants of the invention may be created by site-directedmutagenesis, induced mutation, or may occur as allelic variants(polymorphisms).

As used herein, a “functional fragment” is a regulatory sequence variantformed by one or more deletions from a larger regulatory element. Forexample, the 5′ portion of a promoter up to the TATA box near thetranscription start site can be deleted without abolishing promoteractivity, as described by Opsahl-Sorteberg, et al., (2004) Gene341:49-58. Such fragments should retain promoter activity, particularlythe ability to drive expression in constitutively. Activity can bemeasured by Northern blot analysis, reporter activity measurements whenusing transcriptional fusions, and the like. See, for example, Sambrook,et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.), herein incorporatedby reference.

Functional fragments can be obtained by use of restriction enzymes tocleave the naturally occurring regulatory element nucleotide sequencesdisclosed herein; by synthesizing a nucleotide sequence from thenaturally occurring DNA sequence; or can be obtained through the use ofPCR technology. See, particularly, Mullis, et al., (1987) MethodsEnzymol. 155:335-350 and Erlich, ed. (1989) PCR Technology (StocktonPress, New York).

For example, a routine way to remove part of a DNA sequence is to use anexonuclease in combination with DNA amplification to produceunidirectional nested deletions of double stranded DNA clones. Acommercial kit for this purpose is sold under the trade name Exo-Size™(New England Biolabs, Beverly, Mass.). Briefly, this procedure entailsincubating exonuclease III with DNA to progressively remove nucleotidesin the 3′ to 5′ direction at 5′ overhangs, blunt ends or nicks in theDNA template. However, exonuclease III is unable to remove nucleotidesat 3′, 4-base overhangs. Timed digests of a clone with this enzymeproduces unidirectional nested deletions.

The entire promoter sequence or portions thereof can be used as a probecapable of specifically hybridizing to corresponding promoter sequences.To achieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length and most preferably at least about 20nucleotides in length. Such probes can be used to amplify correspondingpromoter sequences from a chosen organism by the well-known process ofpolymerase chain reaction (PCR). This technique can be used to isolateadditional promoter sequences from a desired organism or as a diagnosticassay to determine the presence of the promoter sequence in an organism.Examples include hybridization screening of plated DNA libraries (eitherplaques or colonies; see, e.g., Innis, et al., (1990) PCR Protocols, AGuide to Methods and Applications, eds., Academic Press).

The constitutive regulatory elements disclosed in the present invention,as well as variants and fragments thereof, are useful in the geneticmanipulation of any plant when operably linked with an isolatednucleotide sequence of interest whose expression is to be controlled toachieve a desired phenotypic response.

By “operably linked” is intended a functional linkage between aregulatory region and a second sequence, wherein the regulatory sequenceinitiates and mediates transcription of the DNA sequence correspondingto the second sequence. The expression cassette will include 5′ and 3′regulatory sequences operably linked to at least one of the sequences ofthe invention.

In one typical embodiment, in the context of an over expressioncassette, operably linked means that the nucleotide sequences beinglinked are contiguous and, where necessary to join two or more proteincoding regions, contiguous and in the same reading frame. In the casewhere an expression cassette contains two or more protein coding regionsjoined in a contiguous manner in the same reading frame, the encodedpolypeptide is herein defined as a “heterologous polypeptide” or a“chimeric polypeptide” or a “fusion polypeptide”. The cassette mayadditionally contain at least one additional coding sequence to beco-transformed into the organism. Alternatively, the additional codingsequence(s) can be provided on multiple expression cassettes.

The regulatory elements of the invention can be operably linked to theisolated nucleotide sequence of interest in any of several ways known toone of skill in the art. The isolated nucleotide sequence of interestcan be inserted into a site within the genome which is 3′ to thepromoter of the invention using site specific integration as describedin U.S. Pat. No. 6,187,994.

The regulatory elements of the invention can be operably linked inexpression cassettes along with isolated nucleotide sequences ofinterest for constitutive expression in the desired plant. Such anexpression cassette is provided with a plurality of restriction sitesfor insertion of the nucleotide sequence of interest under thetranscriptional control of the regulatory elements. Alternatively, aspecific result can be achieved by providing for a reduction ofexpression of one or more endogenous products, particularly enzymes orcofactors in the plant. This down regulation can be achieved throughmany different approaches known to one skilled in the art, includingantisense, cosupression, use of hairpin formations or others anddiscussed infra. Importation or exportation of a cofactor also allowsfor control of plant composition. It is recognized that the regulatoryelements may be used with their native or other coding sequences toincrease or decrease expression of an operably linked sequence in thetransformed plant or seed.

General categories of genes of interest for the purposes of the presentinvention include for example, those genes involved in information, suchas zinc fingers; those involved in communication, such as kinases; andthose involved in housekeeping, such as heat shock proteins. Morespecific categories of transgenes include genes encoding importanttraits for agronomics, insect resistance, disease resistance, herbicideresistance and grain characteristics. Still other categories oftransgenes include genes for inducing expression of exogenous productssuch as enzymes, cofactors and hormones from plants and other eukaryotesas well as prokaryotic organisms.

Modifications that affect grain traits include increasing the content ofoleic acid or altering levels of saturated and unsaturated fatty acids.Likewise, the level of plant proteins, particularly modified proteinsthat improve the nutrient value of the plant, can be increased. This isachieved by the expression of such proteins having enhanced amino acidcontent.

Increasing the levels of lysine and sulfur-containing amino acids may bedesired as well as the modification of starch type and content in theseed. Hordothionin protein modifications are described in WO 9416078filed Apr. 10, 1997; WO 9638562 filed Mar. 26, 1997; WO 9638563 filedMar. 26, 1997 and U.S. Pat. No. 5,703,409 issued Dec. 30, 1997. Anotherexample is lysine and/or sulfur-rich plant protein encoded by thesoybean 2S albumin described in WO 9735023 filed Mar. 20, 1996 and thechymotrypsin inhibitor from barley, Williamson, et al., (1987) Eur. J.Biochem. 165:99-106.

Agronomic traits in plants can be improved by altering expression ofgenes that: affect the response of plant growth and development duringenvironmental stress, Cheikh-N, et al., (1994) Plant Physiol.106(1):45-51 and genes controlling carbohydrate metabolism to reducekernel abortion in maize, Zinselmeier, et al., (1995) Plant Physiol.107(2):385-391.

It is recognized that any gene of interest, including the native codingsequence, can be operably linked to the regulatory elements of theinvention and expressed in the plant.

Commercial traits in plants can be created through the expression ofgenes that alter starch or protein for the production of paper,textiles, ethanol, polymers or other materials with industrial uses.

Means of increasing or inhibiting a protein are well known to oneskilled in the art and, by way of example, may include, transgenicexpression, antisense suppression, co-suppression methods, including butnot limited to: RNA interference, gene activation or suppression usingtranscription factors and/or repressors, mutagenesis includingtransposon tagging, directed and site-specific mutagenesis, chromosomeengineering (see, Nobrega, et. al., (2004) Nature 431:988-993),homologous recombination, TILLING (Targeting Induced Local Lesions InGenomes) and biosynthetic competition to manipulate, the expression ofproteins. Many techniques for gene silencing are well known to one ofskill in the art, including but not limited to knock-outs (such as byinsertion of a transposable element such as Mu, Vicki Chandler, TheMaize Handbook chapter 118 (Springer-Verlag 1994) or other geneticelements such as a FRT, Lox or other site specific integration site; RNAinterference (Napoli, et al., (1990) Plant Cell 2:279-289; U.S. Pat. No.5,034,323, Sharp (1999) Genes Dev. 13:139-141, Zamore, et al., (2000)Cell 101:25-33 and Montgomery, et al., (1998) PNAS USA 95:15502-15507);virus-induced gene silencing (Burton, et al., (2000) Plant Cell12:691-705 and Baulcombe (1999) Curr. Op. Plant Bio. 2:109-113);target-RNA-specific ribozymes (Haseloff, et al., (1988) Nature334:585-591); hairpin structures (Smith, et al., (2000) Nature407:319-320; WO 99/53050 and WO 98/53083); MicroRNA (Aukerman and Sakai,(2003) Plant Cell 15:2730-2741); ribozymes (Steinecke, et al., (1992)EMBO J. 11:1525 and Perriman, et al., (1993) Antisense Res. Dev. 3:253);oligonucleotide mediated targeted modification (e.g., WO 03/076574 andWO 99/25853); zinc-finger targeted molecules (e.g., WO 01/52620; WO03/048345 and WO 00/42219) and other methods or combinations of theabove methods known to those of skill in the art.

Any method of increasing or inhibiting a protein can be used in thepresent invention. Several examples are outlined in more detail belowfor illustrative purposes.

The nucleotide sequence operably linked to the regulatory elementsdisclosed herein can be an antisense sequence for a targeted gene. (See,e.g., Sheehy, et al., (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.5,107,065; 5,453,566 and 5,759,829). By “antisense DNA nucleotidesequence” is intended a sequence that is in inverse orientation to the5′-to-3′ normal orientation of that nucleotide sequence. When deliveredinto a plant cell, expression of the antisense DNA sequence preventsnormal expression of the DNA nucleotide sequence for the targeted gene.The antisense nucleotide sequence encodes an RNA transcript that iscomplementary to and capable of hybridizing with the endogenousmessenger RNA (mRNA) produced by transcription of the DNA nucleotidesequence for the targeted gene. In this case, production of the nativeprotein encoded by the targeted gene is inhibited to achieve a desiredphenotypic response. Thus the regulatory sequences disclosed herein canbe operably linked to antisense DNA sequences to reduce or inhibitexpression of a native protein in the plant.

As noted, other potential approaches to impact expression of proteins inthe plant include traditional co-supression, that is, inhibition ofexpression of an endogenous gene through the expression of an identicalstructural gene or gene fragment introduced through transformation(Goring, et al., (1991) Proc. Natl. Acad Sci. USA 88:1770-1774co-suppression; Taylor, (1997) Plant Cell 9:1245; Jorgensen, (1990)Trends Biotech. 8(12):340-344; Flavell, (1994) PNAS USA 91:3490-3496;Finnegan, et al., (1994) Bio/Technology 12:883-888 and Neuhuber, et al.,(1994) Mol. Gen. Genet. 244:230-241). In one example, co-supression canbe achieved by linking the promoter to a DNA segment such thattranscripts of the segment are produced in the sense orientation andwhere the transcripts have at least 65% sequence identity to transcriptsof the endogenous gene of interest, thereby supressing expression of theendogenous gene in said plant cell. (See, U.S. Pat. No. 5,283,184). Theendogenous gene targeted for co-suppression may be a gene encoding anyprotein that accumulates in the plant species of interest. For example,where the endogenous gene targeted for co-suppression is the 50 kDgamma-zein gene, co-suppression is achieved using an expression cassettecomprising the 50 kD gamma-zein gene sequence or variant or fragmentthereof.

Additional methods of co-suppression are known in the art and can besimilarly applied to the instant invention. These methods involve thesilencing of a targeted gene by spliced hairpin RNA's and similarmethods also called RNA interference and promoter silencing (see, Smith,et al., (2000) Nature 407:319-320, Waterhouse and Helliwell, (2003))Nat. Rev. Genet. 4:29-38; Waterhouse, et al., (1998) Proc. Natl. Acad.Sci. USA 95:13959-13964; Chuang and Meyerowitz, (2000) Proc. Natl. Acad.Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Phystiol.129:1723-1731 and PCT Patent Application Numbers WO 99/53050; WO99/49029; WO 99/61631; WO 00/49035 and U.S. Pat. No. 6,506,559.

For mRNA interference, the expression cassette is designed to express anRNA molecule that is modeled on an endogenous miRNA gene. The miRNA geneencodes an RNA that forms a hairpin structure containing a 22-nucleotidesequence that is complementary to another endogenous gene (targetsequence). miRNA molecules are highly efficient at inhibiting theexpression of endogenous genes and the RNA interference they induce isinherited by subsequent generations of plants.

In one embodiment, the polynucleotide to be introduced into the plantcomprises an inhibitory sequence that encodes a zinc finger protein thatbinds to a gene encoding a protein of the invention resulting in reducedexpression of the gene. In particular embodiments, the zinc fingerprotein binds to a regulatory region of a gene of the invention. Inother embodiments, the zinc finger protein binds to a messenger RNAencoding a protein and prevents its translation. Methods of selectingsites for targeting by zinc finger proteins have been described, forexample, in U.S. Pat. No. 6,453,242 and methods for using zinc fingerproteins to inhibit the expression of genes in plants are described, forexample, in US Patent Publication Number 2003/0037355.

The regulatory region of the invention may also be used in conjunctionwith another promoter. In one embodiment, the plant selection marker andthe gene of interest can be both functionally linked to the samepromoter. In another embodiment, the plant selection marker and the geneof interest can be functionally linked to different promoters. In yetthird and fourth embodiments, the expression vector can contain two ormore genes of interest that can be linked to the same promoter ordifferent promoters. For example, the SB-UBI promoter described here canbe used to drive the gene of interest and the selectable marker, or adifferent promoter used for one or the other. These other promoterelements can be those that are constitutive or sufficient to renderpromoter-dependent gene expression controllable as being cell-typespecific, tissue-specific or time or developmental stage specific orbeing inducible by external signals or agents. Such elements may belocated in the 5′ or 3′ regions of the gene. Although the additionalpromoter may be the endogenous promoter of a structural gene ofinterest, the promoter can also be a foreign regulatory sequence.Promoter elements employed to control expression of product proteins andthe selection gene can be any plant-compatible promoters. These can beplant gene promoters, such as, for example, the ubiquitin promoter (EPPatent Application Number 0 342 926); the promoter for the small subunitof ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO) (Coruzzi, et al.,(1984); Broglie, et al., (1984)) or promoters from the tumor-inducingplasmids from Agrobacterium tumefaciens, such as the nopaline synthase,octopine synthase and mannopine synthase promoters (Velten and Schell,(1985)) that have plant activity; or viral promoters such as thecauliflower mosaic virus (CaMV) 19S and 35S promoters (Guilley, et al.,(1982); Odell, et al., (1985)), the figwort mosaic virus FLt promoter(Maiti, et al., (1997)) or the coat protein promoter of TMV(Grdzelishvili, et al., (2000)).

The range of available plant compatible promoters includes tissuespecific and inducible promoters. An inducible regulatory element is onethat is capable of directly or indirectly activating transcription ofone or more DNA sequences or genes in response to an inducer. In theabsence of an inducer the DNA sequences or genes will not betranscribed. Typically the protein factor that binds specifically to aninducible regulatory element to activate transcription is present in aninactive form which is then directly or indirectly converted to theactive form by the inducer. The inducer can be a chemical agent such asa protein, metabolite, growth regulator, herbicide or phenolic compoundor a physiological stress imposed directly by heat, cold, salt or toxicelements or indirectly through the actin of a pathogen or disease agentsuch as a virus. A plant cell containing an inducible regulatory elementmay be exposed to an inducer by externally applying the inducer to thecell or plant such as by spraying, watering, heating or similar methods.

Any inducible promoter can be used in the instant invention. See, Ward,et al., (1993). Exemplary inducible promoters include ecdysone receptorpromoters, U.S. Pat. No. 6,504,082; promoters from the ACE1 system whichresponds to copper (Mett, et al., (1993)); In2-1 and In2-2 gene frommaize which respond to benzenesulfonamide herbicide safeners (U.S. Pat.No. 5,364,780; the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides; andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena, et al., (1991) and McNellis, et al., (1998) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz, et al., (1991) and U.S. Pat. Nos. 5,814,618 and5,789,156). Alternatively, plant promoters such as heat shock promotersfor example soybean hsp 17.5-E (Gurley, et al., (1986)) orethanol-inducible promoters (Caddick, et al., (1998)) may be used. See,International Patent Application Number WO 91/19806 for a review ofillustrative plant promoters suitably employed in the present invention.

Constitutive promoters can be utilized to target enhanced transcriptionand/or expression within a particular plant tissue. Promoters mayexpress in the tissue of interest, along with expression in other planttissue, may express strongly in the tissue of interest and to a muchlesser degree than other tissue or may express highly preferably in thetissue of interest. Constitutive promoters include those described inYamamoto, et al., (1997); Kawamata, et al., (1997); Hansen, et al.,(1997); Russell, et al., (1997); Rinehart, et al., (1996); Van Camp, etal., (1996); Canevascini, et al., (1996); Yamamoto, et al., (1994); Lam(1994); Orozco, et al., (1993); Matsuoka, et al., (1993) andGuevara-Garcia, et al., (1993).

The expression cassette may also include at the 3′ terminus of theisolated nucleotide sequence of interest, a transcriptional andtranslational termination region functional in plants. The terminationregion can be native with the promoter nucleotide sequence of thepresent invention, can be native with the DNA sequence of interest, orcan be derived from another source. Thus, any convenient terminationregions can be used in conjunction with the promoter of the invention,and are available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See also,Guerineau, et al., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot,(1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev. 5:141-149;Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990)Gene 91:151-158; Ballas, et al., 1989) Nucleic Acids Res. 17:7891-7903;Joshi, et al., (1987) Nucleic Acid Res. 15:9627-9639.

The expression cassettes can additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region),Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130;potyvirus leaders, for example, TEV leader (Tobacco Etch Virus),Allison, et al., (1986); MDMV leader (Maize Dwarf Mosaic Virus),Virology 154:9-20; human immunoglobulin heavy-chain binding protein(BiP), Macejak, et al., (1991) Nature 353:90-94; untranslated leaderfrom the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling,et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV),Gallie, et al., (1989) Molecular Biology of RNA, pages 237-256 and maizechlorotic mottle virus leader (MCMV), Lommel, et al., (1991) Virology81:382-385. See also, Della-Cioppa, et al., (1987) Plant Physiology84:965-968. The cassette can also contain sequences that enhancetranslation and/or mRNA stability such as introns.

In those instances where it is desirable to have an expressed product ofan isolated nucleotide sequence directed to a particular organelle,particularly the plastid, amyloplast or to the endoplasmic reticulum orsecreted at the cell's surface or extracellularly, the expressioncassette can further comprise a coding sequence for a transit peptide.Such transit peptides are well known in the art and include, but are notlimited to: the transit peptide for the acyl carrier protein, the smallsubunit of RUBISCO, plant EPSP synthase and the like.

In preparing the expression cassette, the various DNA fragments can bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers can be employed to join the DNA fragmentsor other manipulations can be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction digests, annealing and resubstitutions such astransitions and transversions, can be involved.

Reporter genes can be included in the transformation vectors. Examplesof suitable reporter genes known in the art can be found in, forexample, Jefferson, et al., (1991) in Plant Molecular Biology Manual,ed. Gelvin, et al., (Kluwer Academic Publishers), pp. 1-33; DeWet, etal., (1987) Mol. Cell. Biol. 7:725-737; Goff, et al., (1990) EMBO J.9:2517-2522; Kain, et al., (1995) BioTechniques 19:650-655 and Chiu, etal., (1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissuescan be included in the transformation vectors. These can include genesthat confer antibiotic resistance or resistance to herbicides. Examplesof suitable selectable marker genes include, but are not limited to:genes encoding resistance to chloramphenicol, Herrera Estrella, et al.,(1983) EMBO J. 2:987-992; methotrexate, Herrera Estrella, et al., (1983)Nature 303:209-213; Meijer, et al., (1991) Plant Mol. Biol. 16:807-820;hygromycin, Waldron, et al., (1985) Plant Mol. Biol. 5:103-108; Zhijian,et al., (1995) Plant Science 108:219-227; streptomycin, Jones, et al.,(1987) Mol. Gen. Genet. 210:86-91; spectinomycin, Bretagne-Sagnard, etal., (1996) Transgenic Res. 5:131-137; bleomycin, Hille, et al., (1990)Plant Mol. Biol. 7:171-176; sulfonamide, Guerineau, et al., (1990) PlantMol. Biol. 15:127-136; bromoxynil, Stalker, et al., (1988) Science242:419-423; glyphosate, Shaw, et al., (1986) Science 233:478-481;phosphinothricin, DeBlock, et al., (1987) EMBO J. 6:2513-2518.

Further, when linking a promoter of the invention with a nucleotidesequence encoding a detectable protein, expression of a linked sequencecan be tracked in the plant, thereby providing a useful so-calledscreenable or scorable markers. The expression of the linked protein canbe detected without the necessity of destroying tissue. More recently,interest has increased in utilization of screenable or scorable markers.By way of example without limitation, the promoter can be linked withdetectable markers including a β-glucuronidase, or uidA gene (GUS),which encodes an enzyme for which various chromogenic substrates areknown (Jefferson, et al., (1986) Proc. Natl. Acad. Sci. USA83:8447-8451); chloramphenicol acetyl transferase; alkaline phosphatase;a R-locus gene, which encodes a product that regulates the production ofanthocyanin pigments (red color) in plant tissues (Dellaporta, et al.,in Chromosome Structure and Function, Kluwer Academic Publishers, Appelsand Gustafson eds., pp. 263-282 (1988); Ludwig, et al., (1990) Science247:449); a p-lactamase gene (Sutcliffe, Proc. Nat'l. Acad. Sci. U.S.A.75:3737 (1978)), which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); a xyIEgene (Zukowsky, et al., Proc. Nat'l. Acad. Sci. U.S.A. 80:1101 (1983)),which encodes a catechol dioxygenase that can convert chromogeniccatechols; an α-amylase gene (Ikuta, et al., Biotech. 8:241 (1990)); atyrosinase gene (Katz, et al., J. Gen. Microbiol. 129:2703 (1983)),which encodes an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone, which in turn condenses to form the easily detectablecompound melanin a green fluorescent protein (GFP) gene (Sheen, et al.,Plant J. 8(5):777-84 (1995)); a lux gene, which encodes a luciferase,the presence of which may be detected using, for example, X-ray film,scintillation counting, fluorescent spectrophotometry, low-light videocameras, photon counting cameras or multiwell luminometry (Teeri, etal., (1989) EMBO J. 8:343); DS-RED EXPRESS (Matz, et al., (1999) NatureBiotech. 17:969-973, Bevis, et al., (2002) Nature Biotech 20:83-87,Haas, et al., (1996) Curr. Biol. 6:315-324); Zoanthus sp. yellowfluorescent protein (ZsYellow) that has been engineered for brighterfluorescence (Matz, et al., (1999) Nature Biotech. 17:969-973, availablefrom BD Biosciences Clontech, Palo Alto, Calif., USA, catalog numberK6100-1); and cyan florescent protein (CYP) (Bolte, et al., (2004) J.Cell Science 117:943-54 and Kato, et al., (2002) Plant Physiol129:913-42).

As noted herein, the present invention provides vectors capable ofexpressing genes of interest under the control of the regulatoryelements. In general, the vectors should be functional in plant cells.At times, it may be preferable to have vectors that are functional in E.coli (e.g., production of protein for raising antibodies, DNA sequenceanalysis, construction of inserts, obtaining quantities of nucleicacids). Vectors and procedures for cloning and expression in E. coli arediscussed in Sambrook, et al., (supra). The transformation vectorcomprising the regulatory sequences of the present invention operablylinked to an isolated nucleotide sequence in an expression cassette, canalso contain at least one additional nucleotide sequence for a gene tobe cotransformed into the organism. Alternatively, the additionalsequence(s) can be provided on another transformation vector. Vectorsthat are functional in plants can be binary plasmids derived fromAgrobacterium. Such vectors are capable of transforming plant cells.These vectors contain left and right border sequences that are requiredfor integration into the host (plant) chromosome. At minimum, betweenthese border sequences is the gene to be expressed under control of theregulatory elements of the present invention. In one embodiment, aselectable marker and a reporter gene are also included.

A transformation vector comprising the particular regulatory sequencesof the present invention, operably linked to an isolated nucleotidesequence of interest in an expression cassette, can be used to transformany plant. In this manner, genetically modified plants, plant cells,plant tissue and the like can be obtained. Transformation protocols canvary depending on the type of plant or plant cell, i.e., monocot ordicot, targeted for transformation. Suitable methods of transformingplant cells include microinjection, Crossway, et al., (1986)Biotechniques 4:320-334; electroporation, Riggs, et al., (1986) Proc.Natl. Acad. Sci. USA 83:5602-5606; Agrobacterium-mediatedtransformation, see, for example, Townsend, et al., U.S. Pat. No.5,563,055; direct gene transfer, Paszkowski, et al., (1984) EMBO J.3:2717-2722 and ballistic particle acceleration, see for example,Sanford, et al., U.S. Pat. No. 4,945,050, Tomes, et al., (1995) in PlantCell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg andPhillips, (Springer-Verlag, Berlin) and McCabe, et al., (1988)Biotechnology 6:923-926. Also see, Weissinger, et al., (1988) AnnualRev. Genet. 22:421-477; Sanford, et al., (1987) Particulate Science andTechnology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol.87:671-674 (soybean); McCabe, et al., (1988) Bio/Technology 6:923-926(soybean); Datta, et al., (1990) Bio/Technology 8:736-740 (rice); Klein,et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein,et al., (1988) Biotechnology 6:559-563 (maize); Klein, et al., (1988)Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990) Biotechnology8:833-839; Hooydaas-Van Slogteren, et al., (1984) Nature (London)311:763-764; Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman, et al., (Longman, N.Y.), pp.197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418and Kaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou, et al., (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens).

The cells that have been transformed can be grown into plants inaccordance with conventional methods. See, for example, McCormick, etal., (1986) Plant Cell Reports 5:81-84. These plants can then be grownand pollinated with the same transformed strain or different strains.The resulting plant having constitutive expression of the desiredphenotypic characteristic can then be identified. Two or moregenerations can be grown to ensure that constitutive expression of thedesired phenotypic characteristic is stably maintained and inherited.

In a further embodiment, plant breeding can be used to introduce thenucleotide sequences into other plants once transformation has occurred.This can be accomplished by any means known in the art for breedingplants such as, for example, cross pollination of the transgenic plantsthat are described above with other plants and selection for plants fromsubsequent generations which express the amino acid sequence. The plantbreeding methods used herein are well known to one skilled in the art.For a discussion of plant breeding techniques, see, Poehlman and Sleper,(1995). Breeding field crops, 4^(th) Edition, Iowa State UniversityPress. Many crop plants useful in this method are bred throughtechniques that take advantage of the plant's method of pollination. Aplant is self-pollinating if pollen from one flower is transferred tothe same or another flower of the same plant. A plant iscross-pollinating if the pollen comes from a flower on a differentplant. For example, in Brassica, the plant is normally self-sterile andcan only be cross-pollinated unless, through discovery of a mutant orthrough genetic intervention, self-compatibility is obtained. Inself-pollinating species, such as rice, oats, wheat, barley, peas,beans, soybeans, tobacco and cotton, the male and female plants areanatomically juxtaposed. During natural pollination, the malereproductive organs of a given flower pollinate the female reproductiveorgans of the same flower. Maize plants (Zea mays L.) can be bred byboth self-pollination and cross-pollination techniques. Maize has maleflowers, located on the tassel, and female flowers, located on the ear,on the same plant. It can self or cross-pollinate.

Pollination can be by any means, including but not limited to hand, windor insect pollination or mechanical contact between the male fertile andmale sterile plant. For production of hybrid seeds on a commercial scalein most plant species pollination by wind or by insects is preferred.Stricter control of the pollination process can be achieved by using avariety of methods to make one plant pool male sterile and the other themale fertile pollen donor. This can be accomplished by hand detassling,cytoplasmic male sterility or control of male sterility through avariety of methods well known to the skilled breeder. Examples of moresophisticated male sterility systems include those described by Brar, etal., U.S. Pat. Nos. 4,654,465 and 4,727,219 and Albertsen, et al., U.S.Pat. Nos. 5,859,341 and 6,013,859.

Backcrossing methods may be used to introduce the gene into the plants.This technique has been used for decades to introduce traits into aplant. An example of a description of this and other plant breedingmethodologies that are well known can be found in references such asPoehlman, et al., (1995). In a typical backcross protocol, the originalvariety of interest (recurrent parent) is crossed to a second variety(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a plantis obtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred gene from thenonrecurrent parent.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Isolation of Regulatory Sequences

Regulatory regions from maize SB-UBI (Sorghum bicolor ubiquitin protein)were isolated from sorghum plants and cloned. Sorghum SB-UBI wasselected as a source of constitutive regulatory elements based on thespatial and temporal expression of its products.

The promoter region consists of a promoter region and an intron whichtogether are an 1899 base pair nucleotide sequence, shown in FIG. 1. Thepromoter is 831 nucleotides, with the intron 1068 nucleotides and whichis underlined. A BLAST of GenBank showed the highest identity was 60% toGenBank accession number DQ225752, G181238239, Upadhyaya, et al.,“Immobile Ac/T-DNA vector pNU400, complete sequence” July 2006.(Dissociation (Ds) constructs, mapped Ds launch pads and atransiently-expressed transposase system suitable for localizedinsertional mutagenesis in rice, Theor. Appl. Genet. 112(7):1326-1341(2006)). The intron had 98% identity to GenBank accession numberAY342494, GI 37912415, Streatfield, et al., “Zea diploperennispolyubiquitin-1 (ubi-1) gene, promoter region and 5′ UTR” August 2004.

Using the maize ubiquitin EST, the sorghum ortholog was identified froman EST collection and used to BLAST a collection of sorghum genomicsequences. Once the corresponding sorghum genomic region was identified,the upstream and downstream sequences were cloned that were similar inlength to the maize ubiquitin promoter, including the intron and theterminator by basing PCR primers on the identified sorghum genomicsequence.

Example 2 Expression Data Using Promoter Sequences

A construct, named PHP28501, was prepared which included the sorghumubiquitin promoter with intron of the invention linked with the DS-REDEXPRESS screenable marker, supra and the pinII or proteinase inhibitorII transcription terminator (An, et al., (1989) Plant Cell 1:115-122).All vectors are constructed using standard molecular biology techniques(Sambrook, et al., supra). Successful subcloning is confirmed byrestriction analysis. Transformation and expression can be confirmed bythe methods outlined in Example 3.

Example 3 Transformation and Regeneration of Maize Callus viaAgrobacterium

Constructs used were as those set forth supra using a binary plasmidwith the left and right borders (see, Hiei, et al., U.S. Pat. No.7,060,876) and the selectable marker for maize-optimized PAT(phosphinothricin acetyl transferase). Jayne, et al., U.S. Pat. No.6,096,947.

Preparation of Agrobacterium Suspension:

Agrobacterium was streaked out from a −80° C. frozen aliquot onto aplate containing PHI-L medium and was cultured at 28° C. in the dark for3 days. PHI-L media comprises 25 ml/l Stock Solution A, 25 ml/l StockSolution B, 450.9 ml/l Stock Solution C and spectinomycin (SigmaChemicals) was added to a concentration of 50 mg/l in sterile ddH₂O(stock solution A: K₂HPO₄ 60.0 g/l, NaH₂PO₄ 20.0 g/l, adjust pH to 7.0w/KOH and autoclaved; stock solution B: NH₄Cl 20.0 g/l, MgSO₄.7H₂O 6.0g/l, KCl 3.0 g/l, CaCl₂ 0.20 g/l, FeSO₄.7H₂O 50.0 mg/l, autoclaved;stock solution C: glucose 5.56 g/l, agar 16.67 g/l (#A-7049, SigmaChemicals, St. Louis, Mo.) and was autoclaved).

The plate can be stored at 4° C. and used usually for about 1 month. Asingle colony was picked from the master plate and was streaked onto aplate containing PHI-M medium [yeast extract (Difco) 5.0 g/l; peptone(Difco) 10.0 g/l; NaCl 5.0 g/l; agar (Difco) 15.0 g/l; pH 6.8,containing 50 mg/L spectinomycin] and was incubated at 28° C. in thedark for 2 days.

Five ml of either PHI-A, [CHU(N6) basal salts (Sigma C-1416) 4.0 g/l,Eriksson's vitamin mix (1000X, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5mg/l (Sigma); 2,4-dichlorophenoxyacetic acid (2,4-D, Sigma) 1.5 mg/l;L-proline (Sigma) 0.69 g/l; sucrose (Mallinckrodt) 68.5 g/l; glucose(Mallinckrodt) 36.0 g/l; pH 5.2] for the PHI basic medium system, orPHI-I [MS salts (GIBCO BRL) 4.3 g/l; nicotinic acid (Sigma) 0.5 mg/l;pyridoxine.HCl (Sigma) 0.5 mg/l; thiamine.HCl 1.0 mg/l; myo-inositol(Sigma) 0.10 g/l; vitamin assay casamino acids (Difco Lab) 1 g/l; 2,4-D1.5 mg/l; sucrose 68.50 g/l; glucose 36.0 g/l; adjust pH to 5.2 w/KOHand filter-sterilize] for the PHI combined medium system and 5 ml of 100mM (3′-5′-Dimethoxy-4′-hydroxyacetophenone, Aldrich chemicals) was addedto a 14 ml Falcon tube in a hood. About 3 full loops (5 mm loop size)Agrobacterium was collected from the plate and suspended in the tube,then the tube vortexed to make an even suspension. One ml of thesuspension was transferred to a spectrophotometer tube and the OD of thesuspension is adjusted to 0.72 at 550 nm by adding either moreAgrobacterium or more of the same suspension medium, for anAgrobacterium concentration of approximately 0.5×109 cfu/ml to 1×109cfu/ml. The final Agrobacterium suspension was aliquoted into 2 mlmicrocentrifuge tubes, each containing 1 ml of the suspension. Thesuspensions were then used as soon as possible.

Embryo Isolation, Infection and Co-Cultivation:

About 2 ml of the same medium (here PHI-A or PHI-I) which is used forthe Agrobacterium suspension was added into a 2 ml microcentrifuge tube.Immature embryos were isolated from a sterilized ear with a sterilespatula (Baxter Scientific Products S1565) and dropped directly into themedium in the tube. A total of about 100 embryos are placed in the tube.The optimal size of the embryos was about 1.0-1.2 mm. The cap was thenclosed on the tube and the tube vortexed with a Vortex Mixer (BaxterScientific Products S8223-1) for 5 sec. at maximum speed. The medium wasremoved and 2 ml of fresh medium were added and the vortexing repeated.All of the medium was drawn off and 1 ml of Agrobacterium suspension wasadded to the embryos and the tube is vortexed for 30 sec. The tube wasallowed to stand for 5 min. in the hood. The suspension of Agrobacteriumand embryos was poured into a Petri plate containing either PHI-B medium[CHU(N6) basal salts (Sigma C-1416) 4.0 g/l; Eriksson's vitamin mix(1000X, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l;L-proline 0.69 g/l; silver nitrate 0.85 mg/l; Gelrite® (Sigma) 3.0 g/l;sucrose 30.0 g/l; acetosyringone 100 mM; pH 5.8], for the PHI basicmedium system, or PHI-J medium [MS Salts 4.3 g/l; nicotinic acid 0.50mg/l; pyridoxine HCl 0.50 mg/l; thiamine.HCl 1.0 mg/l; myo-inositol100.0 mg/l; 2,4-D 1.5 mg/l; sucrose 20.0 g/l; glucose 10.0 g/l;L-proline 0.70 g/l; MES (Sigma) 0.50 g/l; 8.0 g/l agar (Sigma A-7049,purified) and 100 mM acetosyringone with a final pH of 5.8 for the PHIcombined medium system. Any embryos left in the tube were transferred tothe plate using a sterile spatula. The Agrobacterium suspension wasdrawn off and the embryos placed axis side down on the media. The platewas sealed with Parafilm® tape or Pylon Vegetative Combine Tape (productnamed “E.G.CUT” and is available in 18 mm×50 m sections; Kyowa Ltd.,Japan) and was incubated in the dark at 23-25° C. for about 3 days ofco-cultivation.

Resting, Selection and Regeneration Steps:

For the resting step, all of the embryos were transferred to a new platecontaining PHI-C medium [CHU(N6) basal salts (Sigma C-1416) 4.0 g/l;Eriksson's vitamin mix (1000X Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5mg/l; 2.4-D 1.5 mg/l; L-proline 0.69 g/l; sucrose 30.0 g/l; MES buffer(Sigma) 0.5 g/l; agar (Sigma A-7049, purified) 8.0 g/l; silver nitrate0.85 mg/l; carbenicillin 100 mg/l; pH 5.8]. The plate was sealed withParafilm® or Pylon tape and incubated in the dark at 28° C. for 3-5days.

Longer co-cultivation periods may compensate for the absence of aresting step since the resting step, like the co-cultivation step,provides a period of time for the embryo to be cultured in the absenceof a selective agent. Those of ordinary skill in the art can readilytest combinations of co-cultivation and resting times to optimize orimprove the transformation

For selection, all of the embryos were then transferred from the PHI-Cmedium to new plates containing PHI-D medium, as a selection medium,[CHU(N6) basal salts (SIGMA C-1416) 4.0 g/l; Eriksson's vitamin mix(1000X, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l;L-proline 0.69 g/l; sucrose 30.0 g/l; MES buffer 0.5 g/l; agar (SigmaA-7049, purified) 8.0 g/l; silver nitrate 0.85 mg/l; carbenicillin (ICN,Costa Mesa, Calif.) 100 mg/l; bialaphos (Meiji Seika K.K., Tokyo, Japan)1.5 mg/l for the first two weeks followed by 3 mg/l for the remainder ofthe time.; pH 5.8] putting about 20 embryos onto each plate.

The plates were sealed as described above and incubated in the dark at28° C. for the first two weeks of selection. The embryos weretransferred to fresh selection medium at two-week intervals. The tissuewas subcultured by transferring to fresh selection medium for a total ofabout 2 months. The herbicide-resistant calli are then “bulked up” bygrowing on the same medium for another two weeks until the diameter ofthe calli is about 1.5-2 cm.

For regeneration, the calli were then cultured on PHI-E medium [MS salts4.3 g/l; myo-inositol 0.1 g/l; nicotinic acid 0.5 mg/l, thiamine.HCl 0.1mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l, Zeatin 0.5 mg/l,sucrose 60.0 g/l, Agar (Sigma, A-7049) 8.0 g/l, Indoleacetic acid (IAA,Sigma) 1.0 mg/l, Abscisic acid (ABA, Sigma) 0.1 mM, Bialaphos 3 mg/l,carbenicillin 100 mg/l adjusted to pH 5.6] in the dark at 28° C. for 1-3weeks to allow somatic embryos to mature. The calli were then culturedon PHI-F medium (MS salts 4.3 g/l; myo-inositol 0.1 g/l; Thiamine.HCl0.1 mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l, nicotinic acid 0.5mg/l; sucrose 40.0 g/l; Gelrite® 1.5 g/l; pH 5.6] at 25° C. under adaylight schedule of 16 hr light (270 uE m-2sec-1) and 8 hr dark untilshoots and roots are developed. Each small plantlet was then transferredto a 25×150 mm tube containing PHI-F medium and is grown under the sameconditions for approximately another week. The plants were transplantedto pots with soil mixture in a greenhouse. DS-RED EXPRESS events aredetermined at the callus stage or regenerated plant stage.

Ability of the SB-UBI promoter and truncated variant to drive expressionin maize was confirmed by DS-RED EXPRESS detection in plant tissue bythe procedures outlined supra.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. All referencescited are incorporate herein by reference.

1. An isolated regulatory element that drives transcription in aconstitutive manner, wherein the regulatory element comprises thenucleotide sequence of SEQ ID NO:
 1. 2. An expression cassettecomprising a regulatory element operably linked to a nucleotide sequencewherein the regulatory element comprises the nucleotide sequence ofclaim
 1. 3. A plant stably transformed with an expression cassette ofclaim
 2. 4. The plant of claim 3, wherein said plant is a monocot. 5.The plant of claim 3, wherein said monocot is maize, wheat, rice,barley, sorghum or rye.
 6. Seed of the plant of claim 3 wherein the seedcomprises the expression cassette.
 7. A method for selectivelyexpressing a nucleotide sequence in a plant cell, the method comprising:a) transforming a plant cell with an expression cassette, the expressioncassette comprising a regulatory element operably linked to a nucleotidesequence wherein the regulatory element comprises the nucleotidesequence set forth in SEQ ID NO: 1; b) growing the plant cell toselectively express the nucleotide sequence.
 8. The method of claim 7wherein the regulatory element initiates expression of the nucleotidesequence in plant tissue.
 9. The method of claim 7 further comprisingregenerating a stably transformed plant from the plant cell; whereinexpression of the nucleotide sequence alters the phenotype of a plant.10. The plant of claim 9, wherein said plant is a monocot.
 11. The plantof claim 9, wherein said monocot is maize, wheat, rice, barley, sorghumor rye.
 12. Seed of the plant of claim 9 wherein the seed comprises theexpression cassette.